The sugar fructose is one of the sweetest sugars known. It has long been known in the art to produce fructose by the isomerization of the relatively less sweet, and less expensive sugar, glucose, utilizing an enzyme having glucose isomerase activity. Extensive descriptions of the process of enzymatic conversion of glucose to fructose may be found in Hamilton, et al. "Glucose Isomerase a Case Study of Enzyme-Catalysed Process Technology", Immobilized Enzymes in Food and Microbial Processes, Olson et al., Plenum Press, N.Y., (1974), pp. 94-106, 112, 115-137; Antrim, et al.; "Glucose Isomerase Production of High-Fructose Syrups", Applied Biochemistry and Bioengineering, Vol. 2, Academic Press (1979); Chen, et al., "Glucose Isomerase (a Review)", Process Biochem., (1980), pp. 36-41; Nordahl, et al. "Fructose Manufacture from Glucose by Immobilized Glucose Isomerase", Chem. Abstracts, Vol. 82, (1975), Abs. No. 110316h; and Takasaki, "Fructose Production Glucose Isomerase", Chem. Abstracts, Vol. 82, (1975), Abs. No. 110316h; and Takasaki, "Fructose Production by Glucose Isomerase", Chem. Abstracts, Vol. 81, (1974), Abs. No. 7647a.
The amount of fructose which may be obtained from the isomerization reaction of glucose is dependent upon the equilibrium of the reaction. Fructose yields can be substantially improved when the reaction is conducted at temperatures above 50.degree. C., and preferably over 60.degree. C. Unfortunately, the higher temperatures necessary to increase isomerization rate have, in the past, also had the adverse effect of denaturing the glucose isomerase, thus reducing the activity and stability of the enzyme. Therefore, for the use of a glucose isomerase to convert glucose to fructose to be exploited to its full potential, a type of enzyme with high resisitance to thermal denaturation is necessary.
The search for high-yield, thermostable glucose isomerase has been progressing for many years. Among the most common sources of glucose isomerase (also frequently referred to as xylose isomerase, because of the ability to convert xylose to xylulose) are a wide range of microorganisms. Among the organisms which have been demonstrated to be glucose isomerase producers are: Streptomyces flavovirens, Streptomyces achromogenes, Streptomyces echinatus, Streptomyces albus, Streptomyces wedmorensis, Streptomyces phaeochromogenes, Streptomyces bobiliae, Streptomyces olivochromogenes, Streptomyces venezuelae, Aerobacter aerogenes, Aerobacter cloacae, Bacillus coagulans, Bacillus megaterium, Bacillus fructosus, Brevibacterium pentaaminoacidicum, Escherichia intermedia, Leuconostoc mesenteriodes, and Paracolobactrum aerogenoides, and various species of the genera Nocardia, Micromonospora, Microbispora, Microellobospora and Arthrobacter. The thrust of reasearch in this area has been identification of a strain of glucose isomerase producing microorganism which synthesizes a thermally stable enzyme. Among the better known and more successful organisms useful in this respect are Bacillus stearothermophilus (U.S. Pat. No. 3,826,714), Ampulariella sp (U.S. Pat. No. 4,308,349); Streptomyces sp. (U.S. Pat. No. 4,317,883) and Psuedonocardia thermophila (Japanese Pat. No. SHO 49[1974] 30588). Of particular interest to the present invention, however, are members of the genus Arthrobacter.
The use of the genus Arthrobacter for the production of glucose isomerase is well known. U.S. Pat. No. 3,935,068 describes a cell-free enzyme system which is aggregated by the use of flocculation and which is capable of continuous isomerization, Arthrobacter is used as a source of the enzyme to be flocculated, within a temperature range of 50.degree.-90.degree. C. U.S. Pat. No. 3,817,832 relates to a method of isomerizing glucose wherein a glucose solution is passed through a bed of microbial cells in which the glucose isomerase has been fixed or stabilized, within a temperature range of 20.degree. to 80.degree. C. and at a pH between 6 and 9. Arthrobacter is the primary microorganism contemplated in this invention. U.S. Pat. No. 4,304,857 relates to a method of whole cell immobilization whereby wet, glucose isomerase-containing microorganisms, preferably Arthrobacter, are added to fumed silica, the resulting mixture extruded, and the cells dried. The resulting particles may be used for, among other enzymatic transformation reactions, the isomerization of glucose to fructose. U.S. Pat. No. 4,059,489 discloses a process for the continuous production of glucose isomerase by maintenance of a limiting amount of nutrient in the cell culture medium, and the use of the enzyme so produced to isomerase glucose to fructose, at a temperature within the range of 20.degree.-90.degree. C. Arthrobacter is among the preferred organisms for this process.
U.S. Pat. No. 3,645,848 (Reissue Nos. 29,689-29,692) relates to a process of utilizing Arthrobacter for the production of glucose isomerase which is operable at temperature of up to 90.degree. C., at a preferred pH of 8. U.S. Pat. No. 3,821,086 (Reissue Nos. 29,130 and 29,136) discloses an enzymatic process utilizing immobilized microbial cells, the preferred example of which is Arthrobacter.
As noted previously, the desired yields are significantly increased at high temperatures by shifting the equilibrium of the isomerization reaction in favor of fructose, and a glucose isomerase which retains its activity, at very high temperatures is highly desirable. It has now been found that a strain of Arthrobacter, ATCC 21748, produces an enzyme which retains its capacity for isomerization at temperatures over 90.degree. C., a significant improvement over previously known Arthrobacter strains.